U.S. patent number 7,504,203 [Application Number 12/099,631] was granted by the patent office on 2009-03-17 for method for assessing sensitivity of a rice blast fungus to a scytalone dehydratase inhibitor.
This patent grant is currently assigned to Kumiai Chemical Industry Co., Ltd.. Invention is credited to Koichiro Kaku, Kiyoshi Kawai, Kozo Nagayama, Tsutomu Shimizu, Satoshi Watanabe.
United States Patent |
7,504,203 |
Kaku , et al. |
March 17, 2009 |
Method for assessing sensitivity of a rice blast fungus to a
scytalone dehydratase inhibitor
Abstract
The present invention provides a method for assessing
sensitivity of a rice blast fungus to a scytalone dehydratase
inhibitor, comprising the steps of: (a) and (b), which (a)
identifies an amino acid in an amino acid sequence of a scytalone
dehydratase isolated from the rice blast fungus, where the amino
acid corresponds to valine at position 75 of SEQ ID NO: 4; and (b)
assesses the sensitivity of the rice blast fungus to the scytalone
dehydratase inhibitor, where if the amino acid identified in step
(a) is methionine, the sensitivity of the rice blast fungus to The
scytalone dehydratase inhibitor is assessed to be lower than that
of a wild-type rice blast fungus which endogenously produces the
scytalone dehydratase of SEQ ID NO: 4.
Inventors: |
Kaku; Koichiro (Shizuoka,
JP), Watanabe; Satoshi (Shizuoka, JP),
Kawai; Kiyoshi (Shizuoka, JP), Shimizu; Tsutomu
(Shizuoka, JP), Nagayama; Kozo (Shizuoka,
JP) |
Assignee: |
Kumiai Chemical Industry Co.,
Ltd. (Tokyo, JP)
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Family
ID: |
27800264 |
Appl.
No.: |
12/099,631 |
Filed: |
April 8, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20080213788 A1 |
Sep 4, 2008 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10507132 |
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7399625 |
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PCT/JP03/01980 |
Feb 24, 2003 |
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Foreign Application Priority Data
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Mar 12, 2002 [JP] |
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2002-066955 |
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Current U.S.
Class: |
435/4; 435/320.1;
435/410; 530/350; 536/23.1; 536/23.2; 435/69.1; 435/325; 435/252.3;
435/232 |
Current CPC
Class: |
C12Q
1/32 (20130101); C12N 9/88 (20130101); G01N
2500/00 (20130101) |
Current International
Class: |
C12Q
1/527 (20060101); C07H 21/00 (20060101); C07K
14/00 (20060101); C12N 1/21 (20060101); C12N
15/00 (20060101); C12N 5/04 (20060101); C12N
5/10 (20060101); C12N 9/88 (20060101); C12P
21/00 (20060101) |
Field of
Search: |
;435/232,320.1,252.3,325,69.1,410,4 ;530/350 ;536/23.1,23.2 |
Other References
Motoyama, T., et al., cDNA cloning, expression, and mutagenesis of
scytalone dehydratase needed for pathogenicity of the rice blast
fungus, Pyricularia oryzae., Biosci. Biotechnol. Biochem. 1998,
vol. 62, No. 3, pp. 564 to 566. cited by other .
Nakasako, M., et al., Cryogenic X-ray crystal structure analysis
for the complex of scytalone dehydratase of a rice blast fungus and
its tight-binding inhibitor, carpropamid: the structural basis of
tight-binding inhibition., Biochemistry 1998, vol. 37, pp. 9931 to
9939. cited by other .
Meinkoth and Wahl, Current Protocols in Molecular Biology,
Hybridization Analysis of DNA Blots, pp. 2.10.8-2.10.11, 1993.
cited by other .
Branden et al., Introduction to Protein Structure, Garland
Publishing Inc., New York, p. 247, 1991. cited by other .
Seffernick et al., J. Bacteriol. 183(8):2405-2410, 2001. cited by
other .
Witkowski et al., Biochemistry 38:11643-11650, 1999. cited by other
.
Wigley et al., Reprod. Fert. Dev. 6:585-588, 1994. cited by other
.
Cameron, E., Molecular Biotechnology 7:253-265, 1997. cited by
other .
Mullins et al., J. Clin. Invest. 97(7):1557-1560, 1996. cited by
other .
Mullins et al., Hypertension 22(4):630-633, 1993. cited by other
.
Jordan et al., "Tight Binding Inhibitors of Scytalone Dehydratase:
Effects of Site-Directed Mutations", Biochemistry, vol. 39, 2000,
pp. 8593-8602. cited by other.
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Primary Examiner: Ramirez; Delia M
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Parent Case Text
CROSS-REFERENCE
This application is a Divisional of U.S. application Ser. No.
10/507,132, now U.S. Pat. No. 7,399,625, filed on Sep. 10, 2004,
which is the national phase of PCT/JP03/01980 filed on Feb. 24,
2003 which designated the United States and which claims priority
to Japanese Application 2002-66955 filed on Mar. 12, 2002. The
entire contents of the above applications are hereby incorporated
by reference.
Claims
What is claimed is:
1. A method for assessing sensitivity of a rice blast fungus to a
scytalone dehydratase inhibitor comprising the steps of: (a)
identifying an amino acid in the amino acid sequence of a scytalone
dehydratase isolated from the rice blast fungus, wherein said amino
acid corresponds to valine at position 75 of SEQ ID NO: 4; and (b)
assessing sensitivity of the rice blast fungus to the scytalone
dehydratase inhibitor, wherein if the amino acid identified in step
(a) is methionine, the sensitivity of the rice blast fungus to the
scytalone dehydratase inhibitor is assessed to be lower than that
of the wild-type rice blast fungus which endogenously produces the
scytalone dehydratase of SEQ ID NO: 4.
Description
FIELD OF THE INVENTION
The present invention relates to a gene coding for scytalone
dehydratase from a rice blast fungus, which is known as a
pathogenic fungus for rice blast.
BACKGROUND ART
Rice blast caused by rice blast fungi (pyricularia oryzae,
Magnaporthe grisea) is recognized in most countries where rice is
cultivated. In particular, in regions having climates of high
temperature and high humidity (e.g., Japan), rice blast is one of
the most serious diseases in agricultural industry. For high yield
rice cultivation, prevention of and disinfestation for rice blast
are essential. Recently, as an alternative to agents with treatment
effects, box-treatment agents having preventive effects are used
for reducing the labor of farmers in prevention and disinfestation
regarding rice blast fungi. Examples of such agents include
scytalone dehydratase (hereinafter, simply referred to as "SCDH")
inhibitors as typified by carpropamid
((1RS,3SR)-2,2-dichloro-N--((R)-1-(4-chlorophenyl)ethyl)-1-ethyl-3-methyl-
cyclopropanecarboxamide)) (Kurahashi et al., J. Pestic. Sci, 23,
22-28, 1998; Motoyama et al., J. Pestic. Sci, 23, 58-61, 1998).
SCDH is an enzyme that catalyzes the dehydration reaction from
scytalone to 1,3,8-trihydroxynaphtalene (hereinafter, simply
referred to as "1,3,8-THN") in melanin biosynthesis pathways.
When a rice blast fungus ruptures and invades a cuticular membrane
of a rice leaf surface, the concentration of glycerol in the
appressorium, an infection-specific organ, increases up to 80 atm.
In order to enclose the glycerol within the appressorium, the
melanin layer of the cell wall is essential (Kamakura et al.,
KASEAA, 39, 340-347, 2001). Inhibition of melanin biosynthesis
prevents formation of the appressorium. Thus, SCDH inhibitors do
not have a direct fungicidal action, but rather are non-fungicidal
agents that exhibit prevention and disinfestation activities by
suppressing pathogenicity.
An SCDH gene from a filamentous fungus was first elucidated with
Pyricularia oryzae. The nucleotide sequence of this gene was not
available to the public and only the three-dimensional structure of
the SCDH protein was reported (Landquist et al., Structure, 2,
937-944, 1994). Thereafter, an SCDH gene from Colletorichum
lagenarium (Kubo et al., Appl. Environment. Microbiol, 62,
4340-4344, 1996; Accession no. D86079), followed by SCDH genes from
Aspergillus fumigatus (Tsai et al., Mol. Microbiol, 26, 175-183,
1997; Accession no. U95042), Pyricularia oryzae (Motoyama et al.,
Biosci. Biotech. Biochem, 62, 564-566, 1998; Accession no.
AB004741) and Ophiostoma floccosum (Wang et al., Accession no.
AF316575) were reported. A three-dimensional structure of an SCDH
protein bound to carpropamid has also been reported (Nakasako et
al., Biochemistry, 37, 9931-9939, 1998; Wawrzak et al., Proteins:
Struct. Func. Genet, 35, 425-439, 1999).
DISCLOSURE OF INVENTION
Recently, rice blast fungi with decreased sensitivity to SCDH
inhibitors such as carpropamid (hereinafter, referred to as
"resistant rice blast fungi") have been discovered. As described
above, since the SCDH inhibitors such as carpropamid are very
important agents in rice cultivation, it is of the utmost concern
to investigate sensitivity determinant factors in resistant rice
blast fungi and to discover effective methods of prevention and
disinfestation for the resistant rice blast fungi in order to
maintain stable rice cultivation.
However, studies concerning resistant rice blast fungi, such as
elucidation of the sensitivity determinants in the resistant rice
blast fungi or localization of habitats of the resistant rice blast
fungi have hardly been made at present.
In order to achieve the above-described objective, the present
inventor has undertaken intensive research and succeeded in
clarifying the sensitivity determinants in the resistant rice blast
fungi, thereby completing the present invention.
Thus, the present invention encompasses the following. (1) A gene
coding for either one of the following proteins (a) or (b):
(a) a protein consisting of the amino acid sequence shown in SEQ ID
NO:2; or
(b) a protein consisting of an amino acid sequence shown in SEQ ID
NO:2 by deletion substitution or addition of one or more amino
acids, which exhibits scytalone dehydratase activity in the
presence of a scytalone dehydratase inhibitor.
(2) A gene according to (1), wherein the scytalone dehydratase
inhibitor inhibits dehydration reaction from scytalone to
1,3,8-trihydroxynaphtalene in a melanin biosynthesis pathway.
(3) A gene according to (1), wherein the scytalone dehydratase
inhibitor is carpropamid. (4) A scytalone dehydratase encoded by
the gene of (1). (5) A recombinant vector comprising the gene of
(1). (6) A transformant obtained by transformation of the
recombinant vector of (5). (7) A method for assessing sensitivity
of a rice blast fungus to a scytalone dehydratase inhibitor,
comprising the steps of:
(a) identifying an amino acid in an amino acid sequence of
scytalone dehydratase in a subject rice blast fungus, which
corresponds to valine at position 75 in the amino acid sequence
shown in SEQ ID NO: 4; and
(b) assessing sensitivity of the subject rice blast fungus to the
scytalone dehydratase inhibitor based on the results of step (a).
(8) A method for assessing sensitivity according to (7), wherein
when the amino acid identified in step (a) is methionine, the
sensitivity of the subject rice blast fungus to the scytalone
dehydratase inhibitor is assessed to be lower than that of a
wild-type rice blast fungus in step (b). (9) A kit for screening an
inhibitor, comprising the scytalone dehydratase of (4). (10) A kit
for assessing a rice blast fungus resistant to a scytalone
dehydratase inhibitor, comprising a pair of primers designed to
flank a nucleotide sequence coding for an amino acid corresponding
to valine at position 75 in the amino acid sequence shown in SEQ ID
NO: 4. (11) A kit for assessing a rice blast fungus resistant to a
scytalone dehydratase inhibitor, comprising an oligonucleotide
including a nucleotide sequence coding for an amino acid
corresponding to valine at position 75 in the amino acid sequence
shown in SEQ ID NO: 4.
Hereinafter, the present invention will be described in detail.
The gene according to the present invention codes for scytalone
dehydratase (hereinafter, referred to as a "mutant SCDH enzyme")
that exhibits scytalone dehydratase activity in the presence of a
scytalone dehydratase inhibitor (hereinafter, referred to as an
"SCDH inhibitor"). In the following description, scytalone
dehydratase with decreased scytalone dehydratase activity in the
presence of an SCDH inhibitor is simply referred to as an "SCDH
enzyme" or a "wild-type SCDH enzyme."
Examples of the SCDH inhibitor include carpropamid
(2,2-dichloro-N-(1-(4-chlorophenyl)ethyl)-1-ethyl-3-methylcyclopropanecar-
boxamide), fenoxanil
(1-(2,4-dichlorophenyl)oxy-N-(1-cyano-1,2-dimethyl)propylethanecarboxamid-
e), diclocymet
(N-[1-(2,4-dichlorophenyl)ethyl]-1-cyano-2,2-dimethylpropanecarboxamide)
and the like. The SCDH inhibitors are usually used as infection
inhibitors for rice with rice blast fungus to inhibit activity of
the SCDH enzyme. Specifically, the SCDH enzyme catalyzes, in the
melanin biosynthesis pathway shown in FIG. 1, a dehydration
reaction from scytalone to 1,3,8-trihydroxynaphtalene (hereinafter,
simply referred to as "1,3,8-THN") and a dehydration reaction from
vermelone to 1,8-dihydroxynaphtalene.
The SCDH inhibitor inhibits the activity of this SCDH enzyme to
prevent formation of an appressorium in a rice blast fungus,
thereby suppressing pathogenicity to rice. In other words, the SCDH
inhibitor decreases infectivity of the rice blast fungus, and thus
prevents an outbreak of rice blast. A mutant SCDH enzyme, however,
exhibits the above-described enzyme activity even in the presence
of the SCDH inhibitor and thus confers resistance to the SCDH
inhibitor upon rice blast fungi. Accordingly, a rice blast fungus
that expresses a mutant SCDH enzyme (hereinafter, referred to as a
"resistant rice blast fungus" or a "resistant strain") does not
allow inhibition of the melanin biosynthesis even in the presence
of an SCDH inhibitor, and an appressorium can be formed to rupture
and invade a cuticular membrane of the a leaf surface. Thus,
resistant rice blast fungi show high infectivity even in the
presence of the SCDH inhibitors.
Examples of the mutant SCDH enzyme include an enzyme having the
amino acid sequence shown in SEQ ID NO:2. The mutant SCDH enzyme
may have an amino acid sequence similar to SEQ ID NO:2 with one or
more amino acids being deleted, substituted or added, which
exhibits scytalone dehydratase activity in the presence of a
scytalone dehydratase inhibitor. As used herein, the expression
"lone or more" means, for example, 1-30, preferably 1-20, and more
preferably 1-10.
The enzyme activity of a wild-type SCDH enzyme or a mutant SCDH
enzyme can be assessed by determining the dehydration reaction from
scytalone to 1,3,8-THN or the dehydration reaction from vermelone
to 1,8-dihydroxynaphtalene. Specifically, a reaction solution
containing the wild-type SCDH enzyme or the mutant SCDH enzyme and
a substrate (scytalone or vermelone) is used to develop an enzyme
reaction. Then, a decrease in the substrate and/or an increase in
the reaction product (1,3,8-THN or 1,8-dihydroxynaphtalene) is
determined, thereby assessing the enzyme activity of the wild-type
SCDH enzyme or the mutant SCDH enzyme.
Specifically, the enzyme reaction from scytalone to 1,3,8-THN may
be determined spectroscopically. For example, the decrease in
scytalone may be determined according to Motoyama et al., J.
Pestic. Sci, 23, 58-61, 1998.
On the other hand, the increase in 1,3,8-THN may be determined by
UV absorption spectra of the scytalone substrate and the 1,3,8-THN
product at 340-360 nm (as shown in FIG. 2). Although the absorption
of scytalone overlaps with the absorption of 1,3,8-THN at 200-300
nm, the absorption of scytalone at 340-360 nm is negligible. In the
determination method using UV absorption spectra at 340-360 nm, a
rate assay where the enzyme reaction is determined for 100 seconds
is employed to determine the sensitivity of the wild-type SCDH
enzyme or the mutant SCDH enzyme to the SCDH inhibitor.
According to this method, the enzyme reaction is proceeded in a
reaction solution, to which a predetermined concentration of an
SCDH inhibitor (e.g., carpropamid) has been added, to determine UV
absorption spectrum at 340-360 nm, thereby determining a
synthesized amount of the reaction product, 1,3,8-THN. The
determined synthesized amount of 1,3,8-THN is divided by the
synthesized amount of 1,3,8-THN in the absence of the SCDH
inhibitor to obtain an inhibition rate of the SCDH inhibitor at
that concentration. The concentration of the SCDH inhibitor is
varied to determine the inhibition rates of the wild-type and
mutant SCDH enzymes and calculate the I.sub.50 value for each
enzyme. From the Iso value for the wild-type SCDH enzyme and the
I.sub.50 value for the mutant SCDH enzyme, R/S ratio is calculated
to assess the sensitivity of the mutant SCDH enzyme to the SCDH
inhibitor. For example, when the calculated R/S ratio is 2 or
higher, the mutant SCDH enzyme may be defined to have lower
sensitivity to the SCDH inhibitor as compared to that of the
wild-type SCDH enzyme.
Determination of the enzyme activity of the mutant SCDH enzyme is
not limited to the above-described method, and any method may be
applied. The method for determining enzyme activity of the mutant
SCDH enzyme may use, for example, quantification of the enzyme
reaction product, 1,3,8-trihydroxynaphtalene, through HPLC
analysis.
A gene coding for the mutant SCDH enzyme (hereinafter, referred to
as a "mutant SCDH gene") may be obtained from either genome DNA
with introns or cDNA without introns as long as it contains the
nucleotide sequence coding for the above-described mutant SCDH
enzyme.
The mutant SCDH gene can be obtained by PCR using primers designed
based on the cDNA sequence of the SCDH enzyme from rice blast
fungus and genome DNA from a rice blast fungus resistant to the
SCDH inhibitor (hereinafter, referred to as a "resistant rice blast
fungus"). The mutant SCDH gene may also be obtained by RT-PCR using
the above-mentioned primers and mRNA extracted from the resistant
rice blast fungus. The cDNA sequence of the SCDH enzyme from the
rice blast fungus is known and described in Motoyama et al.,
Biosci. Biotech. Biochem, 62, 564-566, 1988 (DNA databank,
Accession no. AB004741).
Examples of the mutant SCDH gene obtained according to such methods
include the nucleotide sequence shown in SEQ ID NO: 1. The results
of comparison between the nucleotide sequence (cDNA) of the mutant
SCDH gene and that of a gene coding for wild-type SCDH enzyme
(hereinafter, referred to as an "SCDH gene") are shown in FIG. 3.
The results of comparison between the nucleotide sequence of the
mutant SCDH gene in genome DNA and that of the SCDH gene are shown
in FIG. 4. As shown in FIGS. 3 and 4, in the mutant SCDH gene, G
(guanosine) at position 223 in the SCDH gene is altered
homozygously by A (adenosine). This alteration means that valine
(Val) at position 75 in the wild-type SCDH enzyme is mutated into
methionine (Met).
As a result of comparing the nucleotide sequence of the mutant SCDH
gene with that of the SCDH gene, T (thymidine) at position 450 was
found to be mutated by C (cytidine) in the mutant SCDH gene.
However, this alteration does not result in amino acid
mutation.
From comparisons in FIGS. 3 and 4, the mutant SCDH gene was found
to have an intron of 81 bases and an intron of approximate 89 bases
between positions 42 and 43 and positions 141 and 142 in the amino
acid sequence of the mutant SCDH enzyme, respectively. Since the
latter intron (located between positions 141 and 142 in the amino
acid sequence of the mutant SCDH enzyme) was followed by poly(A)
strand, and when PCR was carried out, the resultant product had
various lengths, exact length thereof was unable to be determined.
Therefore, it is expressed as "about 89 bases."
The mutant SCDH gene is not limited to the nucleotide sequence
shown in SEQ ID NO: 1, and may be any nucleotide sequence coding
for a protein consisting of the amino acid sequence shown in SEQ ID
NO: 2, or an amino acid sequence shown in SEQ ID NO: 2 by deletion
substitution or addition of one or more amino acids, which exhibits
scytalone dehydratase activity in the presence of a scytalone
dehydratase inhibitor. Examples of such nucleotide sequence include
a nucleotide sequence shown in SEQ ID NO: 1, which includes a
nucleotide substitution that does not result in amino acid
mutation.
The mutant SCDH gene may be a nucleotide sequence coding for a
protein that exhibits scytalone dehydratase activity in the
presence of a scytalone dehydratase inhibitor, and capable of
hybridizing to a nucleotide sequence complementary to the
nucleotide sequence shown in SEQ ID NO: 1 under stringent
conditions. Stringent conditions mean, for example, a sodium
concentration of 10-300 mM, preferably 20-100 mM, and a temperature
of 25-70.degree. C., preferably 42-55.degree. C.
The mutant SCDH gene may be obtained by PCR using, as a template,
genome DNA from a rice blast fungus that infects rice even in the
presence of the SCDH inhibitor and a pair of primers with
predetermined sequences. The genome DNA is prepared according to a
method using CTBA (cetyltrimethylammonium bromide) as an extract
solution, a method via SDS/phenol or phenol/chloroform extraction,
or with a commercially available kit (e.g., the DNeasy Plant System
from Qiagen, the Nucleon PhytoPure kit from Amersham Biosciences,
etc.), although its preparation is not limited to these
methods.
Furthermore, the mutant SCDH gene can be obtained by extracting
total mRNA from a rice blast fungus that infects rice even in the
presence of the SCDH inhibitor and using the total mRNA and a pair
of primers having predetermined sequences in RT-PCR. Total mRNA can
be extracted from a rice blast fungus, for example, by a guanidium
method, an SDS-phenol method, phenol/chloroform extraction with the
RNAeasy Total RNA System from Qiagen, the Quick Prep Micro mRNA
Purification Kit or the Quick Prep Total RNA Extraction Kit from
Amersham Biosciences, although its preparation is not limited to
these methods.
The pair of primers used in the above-described PCR and RT-PCR may
be designed to flank the SCDH gene based on the nucleotide sequence
of genome DNA from, for example, a rice blast fungus deposited with
s gene bank. The pair of primers may also be designed by further
adding a functional sequence based on a nucleotide sequence of
genome DNA from a rice blast fungus. Examples of functional
sequences include a sequence recognized by a restriction enzyme for
linking to a vector, and an insertion sequence for reading frame
adjustment.
Examples of the pair of primers include, but are not limited to,
the following sequences:
TABLE-US-00001 Primer 1 (SEQ ID NO: 5): 5'-GCAGTGATACCCACACCAAAG-3'
Primer 2 (SEQ ID NO: 6): 5'-TTATTTGTCGGCAAAGGTCTCC-3' Primer 3 (SEQ
ID NO: 7): 5'-AGTTCGAACTGGAATTCAACCGGCACGCATGATGCATGCATTTA-3'
Primer 4 (SEQ ID NO: 8): 5'-ATGGGTTCGCAAGTTCAAAAG-3' Primer 5 (SEQ
ID NO: 9): 5'-GTGGCCCTTCATGGTGACCTCCT-3' Primer 6 (SEQ ID NO: 10):
5'-ACAAGCTCTGGGAGGCAATG-3' Primer 7 (SEQ ID NO: 11):
5'-ATCGTCGACGTGAATTCGTGTTGTAAAAGCCGCCAAC-3'
Primers 1, 4, 6 and 7 are sense primers while Primers 2, 3 and 5
are antisense primers. Therefore, one of the pair of primers is
selected from the sense primers and the other is selected from the
antisense primers.
Primer 2 is synthesized based on the nucleotide sequence disclosed
in publication (Motoyama et al., Biosci. Biotech. Biochem, 62,
564-566, 1988), and the underlined base is "G." However, the
corresponding base in Accession no. AB004741 from DNA data bank is
"C." Although the correct base is "C," no effect is caused on the
results from PCR and RT-PCR even when the base is "G." Underlined
letters in Primers 3 and 7 indicate EcoRI recognized sequences.
These EcoRI recognized sequences can be exploited upon
incorporation into a protein expression vector or the like.
Nucleotide sequences 5' to the EcoRI recognized sequences in
Primers 3 and 7 are added to give enough margin for EcoRI to
recognize the EcoRI recognized sequences. In Primer 7, two
nucleotide sequences 3' to the EcoRI recognized sequences (i.e.,
"GT" at positions 18 and 19 in Primer 7) are nucleotides for
allowing reading frame adjustment upon incorporation into a protein
expression vector (pGEX-2T).
For example, RT-PCR is carried out using Primers 7 and 3 with total
RNA as a template. The obtained PCR product is treated with EcoRI,
and then incorporated into pGEX-2T (Amersham Biosciences) that has
been subjected to EcoRI digestion and BAP treatment with alkaline
phosphatase in advance, thereby preparing a plasmid. The plasmid
was deposited with the International Patent Organism Depositary,
National Institute of Advanced Industrial Science and Technology
(Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) on Mar.
8, 2002 under the Budapest Treaty, as Rice Blast Mutant SCDH cDNA
(FERM BP-7948).
This plasmid (Rice Blast Mutant SCDH cDNA) is capable of expressing
an SCDH enzyme as a fusion protein with glutathione-S-transferase
(hereinafter, referred to as "GST") in a host such as E. coli. A
plasmid with a mutant SCDH gene may be constructed to be applicable
to a cell-free protein expressing system.
Furthermore, the mutant SCDH gene may be obtained using a
predetermined probe and a cDNA library from rice blast fungi that
infect rice even in the presence of an SCDH inhibitor.
The mutant SCDH gene may also be obtained by mutagenesis of a
wild-type SCDH gene. For example, a mutant SCDH gene may be
obtained through the so-called site-directed mutagenesis using
primers designed to alter a codon in a wild-type SCDH gene coding
for valine (Val) at position 75 by a codon coding for methionine
(Met). A commercially available kit may be used to obtain a mutant
SCDH gene using the site-directed mutagenesis. Examples of
commercially available kits include the TaKaRa LA PCR in vitro
Mutagenesis kit (Takara).
The above-described mutant SCDH gene is useful for screening a
novel SCDH inhibitor which decreases the infectivity of a resistant
rice blast fungus, as illustrated in the Examples below.
Specifically, an expression vector operatively incorporating the
above-described mutant SCDH gene is used to express the mutant SCDH
enzyme, the enzyme activity of which is in turn determined in the
presence of a candidate agent for a novel SCDH inhibitor. By
determining whether or not the enzyme activity of the mutant SCDH
enzyme is decreased in the presence of the candidate agent, a novel
SCDH inhibitor can be screened.
Specifically, according to conventional determination methods,
inhibition of appressorium formation by a rice blast fungus in the
presence of a candidate agent is assessed by a so-called pot test
or a test based on observation of an appressorium involving the
rupture of cellophane affixed on an agar petri dish, and thus these
methods are hardly capable of rapid screening for an SCDH
inhibitor. On the other hand, according to the above-described
method, enzyme activity of an SCDH enzyme can be measured by a
simple procedure, allowing rapid screening for a novel SCDH
inhibitor.
From the nucleotide sequence analysis of the above-described mutant
SCDH gene, it was found that the mutant SCDH enzyme in which valine
(Val) at position 75 in the SCDH enzyme had been altered by
methionine (Met) exhibited enzyme activity in the presence of the
SCDH inhibitor. Therefore a nucleotide sequence coding for the
amino acid at position 75 in the SCDH enzyme may be analyzed to
determine whether the subject SCDH gene has conferred resistance to
an SCDH inhibitor.
Specifically, when investigating whether or not a rice blast
fungus, for example, from a predetermined region (a subject rice
blast fungus) has sensitivity to an SCDH inhibitor, the amino acid
at position 75 in the SCDH enzyme coded by the SCDH gene (the
subject SCDH gene) from the subject rice blast fungus may be
identified to assess the sensitivity of the subject rice blast
fungus to the SCDH inhibitor.
The nucleotide sequence coding for the amino acid at position 75 in
the subject SCDH enzyme may be identified according to any method
and is not limited to a particular method. In order to sequence the
nucleotide sequence coding for the amino acid at position 75 in the
SCDH enzyme, for example, at least a pair of primers designed to
flank the nucleotide sequence comprising the nucleotide sequence
coding for the amino acid at position 75 in the SCDH enzyme and
template DNA (cDNA or genome DNA) are used to sequence a
predetermined region of the template DNA. Based on the sequenced
nucleotide sequence, the amino acid at position 75 in the subject
SCDH enzyme can be identified.
For sequencing the nucleotide sequence coding for the amino acid at
position 75 in the subject SCDH enzyme, the genome DNA as the
template is preferably obtained through solid cultivation of the
subject rice blast fungus, followed by collection of filamentous
mycelia and microwave irradiation of the mycelia. Irradiation with
microwaves may be carried out, for example, using a microwave oven
or the like. The genome DNA as the template can be obtained in a
short time by this method, as compared to the standard method of
harvesting the subject rice blast fungus after liquid culture and
extracting genome DNA therefrom.
For determining the nucleotide sequence coding for the amino acid
at position 75 in the subject SCDH enzyme, one of the primers is
preferably designed to hybridize near, for example, a location 40
bases upstream from the nucleotide sequence coding for the amino
acid at position 75. Consequently, the nucleotide sequence coding
for the amino acid at position 75 in the subject SCDH enzyme can be
determined in a short time.
Furthermore, for determining the nucleotide sequence coding for the
amino acid at position 75 in the subject SCDH enzyme, an
oligonucleotide comprising a nucleotide sequence coding for an
amino acid corresponding to valine at position 75 in the amino acid
sequence shown in SEQ ID NO: 4 may be used. For example, the
oligonucleotide is designed to hybridize to the gene coding for the
subject SCDH enzyme when the amino acid at position 75 in the
subject SCDH enzyme is methionine. Then, via colony hybridization
or Southern hybridization using this oligonucleotide as a probe,
the amino acid at position 75 in the subject rice blast fungus may
be identified. The sensitivity of the subject rice blast fungus to
an SCDH inhibitor may also be assessed through this method.
Moreover, for analyzing the amino acid at position 75 in the
subject SCDH enzyme, single-stranded DNA conformation polymorphism
(hereinafter, referred to as "SSCP") may be exploited.
Specifically, difference in mobility patterns between a wild-type
SCDH gene and a resistance SCDH gene due to difference in
single-stranded conformation is detected in advance, and compared
to a mobility pattern based on the single-stranded conformation of
the subject SCDH gene. Accordingly, the nucleotide sequence of the
subject SCDH gene coding for the amino acid at position 75 in the
SCDH enzyme can be identified. By exploiting SSCP for analyzing the
amino acid at position 75 in the subject SCDH enzyme, sensitivity
of the subject rice blast fungus to the SCDH inhibitor can be
determined very quickly.
For analyzing the amino acid at position 75 in the subject SCDH
enzyme, modified PCR-restriction fragment length polymorphism
(RFLP) analysis (hereinafter, referred to as "modified PCR-RFLP
method") may also be applied. Specifically, by modified PCR-RFLP
analysis, mutation of valine (Val) at position 75 into methionine
(Met) (hereinafter, referred to as "Val75Met mutation") in the SCDH
enzyme from the subject rice blast fungus can be tested in a simple
manner.
In the modified PCR-RFLP analysis, one of the primers used for PCR
does not comprise the base at position 223 (the base contained in
the codon coding for the amino acid at position 75 in the SCDH
enzyme) and is designed to have a restriction-enzyme-recognized
sequence at the 3'-end depending upon the type of the base at
position 223. This primer may contain one or more bases partially
mismatching the nucleotide sequence of the genome DNA or cDNA as
the template, while containing the above
restriction-enzyme-recognized sequence. The
restriction-enzyme-recognized sequence is not particularly limited
and may be a sequence recognized by XbaI.
According to the modified PCR-RFLP analysis, first, PCR is
performed using a pair of primers designed as described above and
genome DNA or cDNA as a template. Upon PCR, various conditions such
as temperature or time may appropriately be determined so that the
desired region of the template can be amplified even if a primer
including one or more bases mismatching the template is used. The
product resulting from PCR contains a restriction-enzyme-recognized
sequence as well as the above-described primer depending on the
base at position 223. The restriction-enzyme-recognized sequence
may not be contained depending on the base at position 223.
Next, the product resulting from PCR is treated with a restriction
enzyme that recognizes the restriction-enzyme-recognized sequence
contained in the above-described primer. The fragments obtained
through this restriction enzyme treatment have different lengths
due to the difference of the base at position 223. Then, the
lengths of the fragments obtained by the restriction enzyme
treatment may be detected, for example, by a method such as
electrophoresis to identify the base at position 223 to analyze the
amino acid at position 75 in the subject SCDH enzyme.
Furthermore, for analyzing the amino acid at position 75 in the
subject SCDH enzyme, a generally known single nucleotide
polymorphism typing method may be employed. Examples of the single
nucleotide polymorphism typing method include the SNaPshot
Multiplex Kit from Applied Biosystems (single primer extension
reaction), the Masscode system from Qiagen (mass spectrometry), the
MassARRAY system from Sequenom, the UCAN method from Takara, the
Invader assay using Cleavase and a method using a microarray.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating a melanin biosynthesis pathway in
a rice blast fungus.
FIG. 2 is a characteristic diagram showing UV absorption spectra of
scytalone and 1,3,8-THN.
FIG. 3 shows comparison between the nucleotide sequence of the SCDH
gene from the rice blast fungus registered with the gene bank (SEQ
ID NO: 13), a nucleotide sequence (cDNA) of an SCDH gene from a
standard strain (SEQ ID NO: 14)and a nucleotide sequence (cDNA) of
an SCDH gene from a resistant strain (SEQ ID NO: 15).
FIG. 4 shows comparison between the nucleotide sequence of the SCDH
gene from the rice blast fungus registered with the gene bank (SEQ
ID NO: 16), a nucleotide sequence (genome DNA) of an SCDH gene from
a standard strain (SEQ ID NO: 17)and a nucleotide sequence (genome
DNA) of an SCDH gene from a resistant strain (SEQ ID NO: 18).
FIG. 5 is a characteristic diagram showing the relationship between
the carpropamid concentrations and the inhibition rates for SCDH
enzyme activity in crude enzyme solutions extracted from the
standard strain and the resistant strains (A and B). In this
diagram, the open circles represent the results for the crude
enzyme solution from the standard strain, the open triangles
represent the results for the crude enzyme solution from the
resistant strain A and the open squares represent the results for
the crude enzyme solution from the resistant strain B.
FIG. 6 are electrophoresis pictures showing the results from the
single-stranded DNA conformation polymorphism (SSCP) analysis
conducted in Example 4, where A (left) shows the results from
electrophoresis without purification using GFX PCR DNA and Gel Band
Purification Kit while B (right) shows the results from
electrophoresis following purification using GFX PCR DNA and Gel
Band Purification Kit.
FIG. 7 is a schematic view showing a method for preparing plasmid
Rice Blast wild SCDH cDNA and Rice Blast Mutant SCDH cDNA. Peptide
sequence is SEQ ID NO: 19.
FIG. 8 is a characteristic diagram showing the relationship between
the carpropamid concentrations and the inhibition rates for SCDH
enzyme activity for the GST-fused SCDH enzyme obtained by
expressing cDNA from the standard strain in E. coli, the GST-fused
SCDH enzyme obtained by expressing cDNA from the resistant strain
in E. coli, the crude enzyme solution from the standard strain and
the crude enzyme solution from the resistant strain. In this
diagram, the open circles represent the results for the crude
enzyme solution from the standard strain, the open triangles
represent the results for the GST-fused SCDH enzyme expressed from
the standard strain cDNA, the closed circles represent the results
for the crude enzyme solution from the resistant strain and the
closed triangles represent the results for the GST-fused SCDH
enzyme expressed from the resistant strain cDNA.
FIG. 9 is a characteristic diagram showing the relationship between
the fenoxanil or diclocymet concentrations and the inhibition rates
for the GST-fused SCDH enzyme obtained by expressing cDNA from the
standard strain in E. coli and the GST-fused SCDH enzyme obtained
by expressing cDNA from the resistant strain in E. coli. In this
diagram, the open circles represent the inhibition of the GST-fused
SCDH enzyme expressed from the standard strain cDNA by fenoxanil,
the open triangles represent the inhibition of the GST-fused SCDH
enzyme expressed from the resistant strain cDNA by fenoxanil, the
closed circles represent the inhibition of the GST-fused SCDH
enzyme expressed from the standard strain cDNA by diclocymet, and
the closed triangles represent the inhibition of the GST-fused SCDH
enzyme expressed from the resistant strain cDNA by diclocymet.
FIG. 10 is an electrophoresis (3% agarose gel) picture showing the
results obtained by analyzing Val75Met mutation in the SCDH enzyme
by applying PCR-RFLP method performed in Example 6.
BEST MODE FOR CARRYING OUT THE INVENTION
EXAMPLES
Hereinafter, the present invention will be described in more detail
by means of examples. The technical scope of the present invention,
however, is not limited by these examples.
Example 1
According to this example, first, filamentous mycelia used for
extracting SCDH enzymes were prepared. Spore solutions
(10.sup.5/ml) containing a rice blast fungus (Pyricularia oryzae)
as a standard (wild-type) strain and carpropamid-resistant rice
blast fungi (resistant strains A and B) were individually added to
200 ml YGPCa liquid culture solutions (pH 6.5) each containing
yeast extract (5 g), glucose (20 g), KH.sub.2PO.sub.4 (0.5 g),
Na.sub.2HPO.sub.4 (0.5 g) and CaCl.sub.2 (0.5 mg), and were grown
at 27.degree. C. for 4 to 5 days.
After the cultivation, the filamentous mycelia were collected
through centrifugation of the culture solutions and washed with
distilled water. Cold acetone, which has five times the weight of
the mycelia, was added, and the results were homogenized with a
Waring blender. The homogenates were centrifuged (15,000.times.g,
20 min.). The precipitates were dried at 4.degree. C. to obtain
acetone powders, which were stored at -85.degree. C.
The obtained acetone powders were used to prepare crude enzyme
solutions containing the SCDH enzymes in order to determine their
enzyme activities. In order to prepare these crude enzyme
solutions, each the acetone powder was suspended in 20 ml of 1/15 M
potassium phosphate buffer (pH 6.8), agitated for 30 minutes while
being iced and then centrifuged at 15,000.times.g for 15 minutes.
Supernatants obtained by centrifugation were used as the crude
enzyme solutions.
Next, to determine the enzyme activities of the SCDH enzymes using
the crude enzyme solutions, first, 1,300 .mu.l of 100 mM phosphate
buffer (pH 6.8) containing 1 mM EDTA, 30 .mu.l of 20 mM scytalone
(ethanol solution), 30 .mu.l ethanol solution of carpropamid at an
appropriate concentration and 1,440 .mu.l ultrapure water were
mixed and pre-incubated at 27.degree. C. for 2 minutes. Then, 200
.mu.l of the crude enzyme solution was added to initiate enzyme
reaction. The amount of 1,3,8-THN produced from scytalone through
enzyme reaction was monitored for 100 seconds as an increase in the
absorbance at UV 350 nm, thereby determining enzyme activity caused
by the SCDH enzyme contained in the crude enzyme solution. The
scytalone substrate was prepared from mycelium obtained through
liquid cultivation of the standard (wild-type) strain in the
presence of carpropamid, according to a routine technique
(Kurahashi et al., J. Pestic. Sci, 23, 22-28, 1998).
The results are shown in FIG. 5. These results were used to
calculate 50% inhibition concentrations (150 values) by probit
analysis. As a result, the 15 value of the crude enzyme solution
extracted from the standard (wild-type) strain with respect to
carpropamid was 7.45 nM while those of the resistant strains A and
B were 163 nM and 157 nM, respectively. From these values, the R/S
ratio was about 21.5. This suggested that a factor for carpropamid
resistance in the resistant strains A and B was the decrease in the
sensitivity of scytalone dehydratase, which is the target of the
carpropamid.
Example 2
In this example, first, filamentous mycelia were prepared as
described below for extracting genome DNA and mRNA from rice blast
fungi. First, a standard (wild-type) strain and
carpropamid-resistant rice blast fungi (resistant strains A and B)
were individually cultured on oatmeal media. After the cultivation,
the mycelium parts were each added to 20 ml potato-dextrose (PD)
liquid media and pre-cultured at 28.degree. C. for 3 days. Since
the pre-cultured filamentous mycelia form themselves into lumps,
they were homogenized with a sterilized Waring blender and 1 ml of
each sample was cultured in a 20 ml PD liquid medium for another
3-5 days. The mycelia were separated by filtration under reduced
pressure and washed with distilled water. These mycelia were ground
in liquid nitrogen using a mortar. The ground powders were stored
at -85.degree. C. Thus, powders from the standard (wild-type)
strain, the resistant strain A and the resistant strain B were
obtained.
For extracting total RNA using the powder from the resistant strain
A, the Rneasy Plant Mini Kit (Qiagen) was used according to the
attached protocol. For extracting genome DNA using the obtained
powder, the Dneasy Plant Mini Kit (Qiagen) was used according to
the attached protocol. The RNA concentration was quantified by
determining the absorption at OD.sub.260 with a spectrophotometer.
DNA concentration was determined by observation of the brightness
on 1% agarose gel or by measurements of the fluorescence spectrum
using Hoe 33258 (Hoechst).
Next, the obtained total RNA was used to prepare cDNA containing a
mutant SCDH gene from the resistant strain. In order to prepare
cDNA containing the mutant SCDH gene, first, the obtained RNA (2
.mu.g) was mixed with 2 .mu.l oligo(dT).sub.20 (10 pmol/.mu.l), 2
.mu.l each of Primer 1 (5'-GCAGTGATACCCACACCAAAG-3', 25 pmol/.mu.l)
(SEQ ID NO: 5)and Primer 2 (5'-TTATTTGTCGGCAAAGGTCTCC-3', 25
pmol/.mu.l) (SEQ ID NO: 6)and RT-PCR beads (Amersham Biosciences)
to a final volume of 50 .mu.l to prepare a reaction solution. The
reaction took place under the following conditions. For cDNA
synthesis, reaction was performed at 42.degree. C. for 30 minutes,
followed by reaction at 95.degree. C. for 30 minutes. Subsequently,
for PCR reaction using the synthesized cDNA as a template, 35
cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 1 minute
and 72.degree. C. for 1 minute were repeated. After the final cycle
at 72.degree. C. for 7 minutes, the reaction was carried out and
terminated. The reaction solution obtained was purified after the
reaction using the GFX PCR DNA and the Gel Band Purification Kit
(Amersham Biosciences) to obtain the RT-PCR product. cDNA
containing the SCDH gene from the standard strain and cDNA
containing the mutant SCDH gene from the resistant strain B were
also obtained in manners similar to the above-described method.
In addition, DNA containing the mutant SCDH gene from the resistant
strain A was prepared using the obtained genome DNA. For preparing
this DNA, first, 4 .mu.l of the obtained genome DNA was mixed with
1 .mu.l each of Primer 1 (5'-GCAGTGATACCCACACCAAAG-3', 25
pmol/.mu.l) (SEQ ID NO: 5) and Primer 3
(5'-AGTTCGAACTGGAATTCAACCGGCACGCATGATGCATGCATTTA-3', 25 pmol/.mu.l)
(SEQ ID NO: 7) and PCR beads (Amersham Biosciences) to a final
volume of 25 .mu.l to prepare a reaction solution. The reaction
took place under the following conditions. For PCR reaction using
the genome DNA as a template, 40 cycles of 95.degree. C. for 30
seconds, 55.degree. C. for 1 minute and 72.degree. C. for 2 minutes
were repeated. After the final cycle at 72.degree. C. for 7
minutes, the reaction was carried out and terminated. The reaction
solution obtained was purified after the reaction using the GFX PCR
DNA and the Gel Band Purification Kit (Amersham Biosciences) to
obtain the PCR product. DNA containing the SCDH gene from the
standard strain and DNA containing the mutant SCDH gene from the
resistant strain B were also obtained in manners similar to the
above-described method.
Then, the obtained RT-PCR product and the PCR product were used to
sequence the nucleotide sequence of cDNA containing the mutant SCDH
gene and the nucleotide sequence of DNA containing the mutant SCDH
gene. Sequencing was performed using the BigDye Terminator Cycle
Sequencing FS Ready Reaction Kit from Applied Biosystems.
The sequencing reaction using this kit was performed in a reaction
solution (total amount: 20 .mu.l) of a mixture of the RT-PCR or the
PCR product as a template, 3.2 .mu.mol primers (Primers 1, 3, 5 and
6) and 8 .mu.l of terminator pre-mix. As the reaction conditions,
40 cycles of 96.degree. C. for 10 seconds, 50.degree. C. for 5
seconds and 60.degree. C. for 4 minutes were repeated. After the
final cycle, the reaction was terminated at 60.degree. C. for 7
minutes. After the reaction, the components such as the die
terminator remaining in the reaction solution were removed by gel
filtration using Auto Seq G-50 (Amersham Bioscience). Then, the
reaction product was analyzed using ABI 310 Genetic Analyzer from
Applied Biosystems for nucleotide sequencing. The nucleotide
sequence of the mutant SCDH gene sequenced using the RT-PCR product
as the template is shown in SEQ ID NO: 1 and the amino acid
sequence of the mutant SCDH enzyme encoded by the mutant SCDH gene
is shown in SEQ ID NO: 2.
The results from the analysis of cDNA of the mutant SCDH gene using
the RT-PCR product as the template are shown in FIG. 3. FIG. 3
shows comparison between the nucleotide sequence of the SCDH gene
from the rice blast fungus registered with the gene bank (Accession
no. A13004741, upper row), the nucleotide sequence of the SCDH gene
analyzed using the RT-PCR product obtained from the standard strain
(middle row) and the nucleotide sequence of the mutant SCDH gene
analyzed using the RT-PCR product obtained from the resistant
strain A (bottom row).
The results from analysis of the mutant SCDH gene present in the
genome DNA using the PCR product as the template are shown in FIG.
4. FIG. 4 shows comparison between the nucleotide sequence of the
SCDH gene from the rice blast fungus registered with the gene bank
(Accession no. AB004741, upper row), the nucleotide sequence of the
SCDH gene analyzed using the PCR product obtained from the standard
strain (middle row) and the nucleotide sequence of the mutant SCDH
gene analyzed using the PCR product obtained from the resistant
strain A (bottom row).
Referring to FIGS. 3 and 4, G (guanosine) at position 223 in the
cDNA nucleotide sequence of the SCDH gene was found to have altered
homozygously by A (adenosine) in the resistant strain A. This means
that valine (Val) at position 75 in the amino acid sequence of the
SCDH enzyme from the standard strain will be mutated into
methionine (Met). The base at position 450 in the cDNA nucleotide
sequence was T (thymidine) in the registered nucleotide sequence
(Accession no. AB004741, upper row in FIG. 3) while it was C
(cytidine) in the standard strain and the resistant strain.
However, since the alteration of the base at position 450 in these
cDNA nucleotide sequences is not associated with amino acid
mutation, it presumably has nothing to do with sensitivity to SCDH
inhibitors.
From FIG. 4, introns with lengths of 81 bases and about 89 bases
were confirmed between positions 42 and 43 and positions 141 and
142, respectively, in the nucleotide sequence shown in SEQ ID NO:
3. Since the latter intron was followed by poly(A) strand and the
products resulting from PCR had various lengths, the exact length
thereof was unable to be determined. Accordingly, it is expressed
as "about 89 bases."
Example 3
A simple assay of mutation of valine (Val) into methionine (Met) at
position 75 (hereinafter, referred to as "Val75Met mutation") in
the SCDH enzymes from rice blast fungi was considered.
A rice blast fungus grown on an oatmeal medium (5% oatmeal, 2%
sucrose and 1.5% agar) at 28.degree. C. was pricked with a
toothpick and transferred into a 1.5 .mu.l microtube. The microtube
was covered with a lid and irradiated with microwave in a microwave
oven (600 W) for 5-7 minutes. Due to this treatment, the cell wall
of the fungus was ruptured.
Next, 50 .mu.l TE buffer (pH 8.0) was added to the microtube, and
the resultant was thoroughly agitated and centrifuged at 14,000 rpm
for 10 minutes. The supernatant containing free genome DNA was
transferred to another microtube and stored at -20.degree. C. One
to five .mu.l of the supernatant was mixed with 1 .mu.l each of
Primer 4 (5'-ATGGGTTCGCAAGTTCAAAAG-3', 25 pmol/.mu.l), (SEQ ID NO:
8) Primer 5 (5'-GTGGCCCTTCATGGTGACCTCCT-3', 25 pmol/.mu.l) (SEQ ID
NO: 9) and PCR beads (Amersham Biosciences) for a final volume of
25 .mu.l to prepare a reaction solution. For PCR reaction, 40
cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 1 minute
and 72.degree. C. for 2 minutes were repeated. After the final
cycle at 72.degree. C. for 7 minutes, the reaction was carried out
and terminated. The reaction solution was purified using the
Invisorb Spin PCRapid Kit (Invitek) to obtain a PCR product. The
PCR product contained in the reaction solution was subjected to
sequencing reaction using the BigDye Terminator Cycle Sequencing FS
Ready Reaction Kit from Applied Biosystems.
For the sequencing reaction, the PCR product as a template, 3.2
.mu.mol of Primer 6 (5'-ACAAGCTCTGGGAGGCAATG-3') (SEQ ID NO: 10)
and 8 .mu.l of terminator pre-mix were mixed to prepare a reaction
solution for a total amount of 20 .mu.l. For the sequencing
reaction, 40 cycles of 96.degree. C. for 10 seconds, 50.degree. C.
for 5 seconds and 60.degree. C. for 4 minutes were repeated. After
the final cycle at 60.degree. C. for 7 minutes, the reaction was
carried out and terminated. After the reaction, components such as
the die terminator remaining in the reaction solution were removed
by gel filtration using the Auto Seq G-50 (Amersham Bioscience).
Then, the reaction product was subjected to sequence analysis using
the ABI 310 Genetic Analyzer from Amersham Biosciences. By using a
47 cm.times.50 .mu.m short capillary column from Amersham
Biosciences, mutation of the amino acid valine at position 75 into
methionine was confirmed in a short time of about 35 minutes per
sample.
Example 4
A simple assay of mutation of valine (Val) into methionine (Met) at
position 75 (hereinafter, referred to as Val75Met mutation) in an
SCDH enzyme from a rice blast fungus was considered by applying a
single-stranded DNA conformation polymorphism (SSCP) analysis.
As in Example 3, a genome DNA solution was simply prepared by
irradiating rice blast fungus filamentous mycelium with microwaves.
Five .mu.l of this genome DNA solution were mixed with 1 .mu.l each
of Primer 6 (5'-ACAAGCTCTGGGAGGCAATG-3', 25 pmol/.mu.l) (SEQ ID NO:
10), Primer 5 (5'-GTGGCCCTTCATGGTGACCTCCT-3', 25 pmol/.mu.l) (SEQ
ID NO: 9)and PCR bead (Amersham Biosciences) for a final volume of
25 .mu.l to prepare a reaction solution. For PCR reaction, 40
cycles of 95.degree. C. for 30 seconds, 55.degree. C. for 1 minute
and 72.degree. C. for 2 minutes were repeated. After the final
cycle, the reaction was terminated at 72.degree. C. for 7 minutes.
As a result of this reaction, 215 bp PCR product was obtained. The
components such as taq DNA polymerase and primers remaining in the
reaction solution were removed using GFX PCR DNA and the Gel Band
Purification Kit (Amersham Biosciences).
Thereafter, a mixture of 0.4 ml of 0.5 M EDTA (pH 8.0), 10 mg of
bromophenol blue and 10 ml of formamide was prepared as a loading
buffer for SSCP. The reaction solution and the loading buffer were
mixed at a ratio of 1:1, heated at 85.degree. C. for 15 minutes and
cooled at once. As a result, the PCR product contained in the
reaction solution became single-stranded DNA.
Then, the mixture of the reaction solution and the loading buffer
were used to perform electrophoresis with the PhastSystem full
automatic electrophoresis system from Amersham Biosciences.
PhastGel Homogeneous 12.5 and PhastGel Native Buffer Strips from
Amersham Biosciences were used as a gel carrier and a buffer
reagent, respectively, for pre-electrophoresis at 400 V, 10 mA, 2.5
W, 4.degree. C., and 100 Vh and for actual electrophoresis at 400
V, 10 mA, 2.5 W, 4.degree. C., and 200 Vh. The results are shown in
FIGS. 6A and 6B. FIG. 6A shows the results from electrophoresis
without the above-described purification using GFX PCR DNA and the
Gel Band Purification Kit. FIG. 6B shows the results from
electrophoresis following the above-described purification using
GFX PCR DNA and the Gel Band Purification Kit.
The electrophoresis patterns of the single stranded DNA are
different in FIGS. 6A and 6B, presumably due to buffer compositions
in the PCR solutions. In any case, difference in the
electrophoresis patterns between the standard strain and the
carpropamid-resistant strains was observed and distinguishable from
FIGS. 6A and 6B.
Example 5
An expression vector incorporating the mutant SCDH gene was
constructed to study its resistance to an SCDH inhibitor.
In order to incorporate a scytalone dehydratase gene from a rice
blast fungus into a protein expression vector pGEX-2T (Amersham
Biosciences), RT-PCR was conducted using Primer 7
(5'-ATCGTCGACGTGAATTCGTCTTGTAAAAGCCGCCAAC-3') (SEQ ID NO: 11) and
Primer 3 (5'-AGTTCGAACTGGAATTCAACCGGCACGCATGATGCATGCATTTA-3') (SEQ
ID NO: 7) having EcoRI cleavage sites at their terminals. The
RT-PCR was conducted according to the method described in Example
1. Primers 7 and 3 were located upstream and downstream from the
open reading frame (ORF) of the SCDH gene, respectively, so as to
flank the whole coding region for the SCDH enzyme.
For RT-PCR, first, total RNA (2 .mu.g each) extracted from the
standard (wild-type) fungus or the carpropamid-resistant rice blast
fungus were mixed with 2 .mu.l oligo(dT).sub.20 (10 pmol/.mu.l), 2
.mu.l each of Primer 4 (25 pmol/.mu.l) and Primer 3 (25 pmol/.mu.l)
and RT-PCR bead (Amersham Biosciences) to prepare a reaction
solution for a final volume of 50 .mu.l. The reaction took place
under the following conditions. For cDNA strand synthesis, the
reaction solutions were reacted at 42.degree. C. for 30 minutes,
followed by reaction at 95.degree. C. for 30 minutes. Subsequently,
PCR reaction was performed by repeating 25 cycles of 95.degree. C.
for 30 seconds, 55.degree. C. for 1 minute and 72.degree. C. for 1
minute. After the reaction, RT-PCR products were purified from the
reaction solutions using GFX PCR DNA and the Gel Band Purification
Kit (Amersham Biosciences) and then eluted with a final volume of
50 .mu.l of sterilized water.
Next, 30 .mu.l of the solution containing one of the RT-PCR
products was mixed with 4 .mu.l of 10.times.H buffer (Takara), 1
.mu.l of EcoRI (12 u/.mu.l, Takara) for a final volume of 40 .mu.l
and subjected to restriction enzyme reaction at 37.degree. C. for 2
hours. After the restriction enzyme reaction, the reaction
solutions were purified with GFX PCR DNA and the Gel Band
Purification Kit (Amersham Biosciences) and eluted with 30 .mu.l
sterilized water.
In addition, 1 .mu.g of GST-fused protein expression vector pGEX-2T
(Amersham Biosciences) was mixed with 1 .mu.l of 10.times.H buffer
(Takara) and 1 .mu.l of EcoRI (12 u/.mu.l, Takara) for a final
volume of 10 .mu.l and subjected to a restriction enzyme reaction
at 37.degree. C. for 1 hour. To this reaction solution, 10 .mu.l of
BAP buffer (TOYOBO), 2.5 .mu.l of alkaline phosphatase (0.4
u/.mu.l, BAP-101, TOYOBO) and 77.5 .mu.l of sterilized water were
added. The resultant was subjected to dephosphorylation reaction at
37.degree. C. for 2 hours.
Then, reaction solutions were prepared by mixing 2 .mu.l of the
EcoRI-digested RT-PCR product, 1 .mu.l of EcoRI/BAP-treated
pGEX-2T, 2 .mu.l sterilized water and 5 .mu.l ligation buffer I
(Ver. 2, Takara) and subjected to ligation reaction at 16.degree.
C. for 12 hours. After the reaction, by following the protocol
attached to the competent cell of E. coli (strain JM109) (Takara),
the reaction solutions were used to transform E. coli JM109. Then,
the transformed E. coli JM109 were spread over LB solid media each
containing 50 ppm ampicillin and subjected to static culture at
37.degree. C. for 12 hours. After the cultivation, a few single
colonies were scraped to perform direct colony PCR. As a result of
the direct colony PCR, pGEX-2T inserted with the SCDH gene in the
direction of interest were screened. The nucleotide sequence was
further sequenced to confirm that the nucleotide sequence of the
inserted SCDH gene was correct. This method is schematically
illustrated in FIG. 7. The plasmid obtained according to this
method was deposited with the International Patent Organism
Depositary, National Institute of Advanced Industrial Science and
Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki,
Japan) on Mar. 8, 2002 under the Budapest Treaty, as Rice Blast
Mutant SCDH cDNA (FERM BP-7948).
Then, E. coli transformed with a pGEX-2T vector containing the
correctly inserted SCDH gene was cultured at 27.degree. C. in 200
ml LB liquid medium containing 50 ppm ampicillin until OD.sub.260
became 0.6-1.0. Thereafter, isopropyl-1-thio-.beta.-D-galactoside
(IPTG) was added to a final concentration of 1 mM and further
subjected to thorough agitation culture at 27.degree. C. for 5
hours. After the cultivation, E. coli was collected by
centrifugation (10,000.times.g, 10 minutes, 4.degree. C.). E. coli
was once suspended in 10 ml of cold 1/15 M potassium phosphate
buffer (pH 6.8) for washing, and then collected by another
centrifugation (10,000.times.g, 10 minutes, 4.degree. C.).
Subsequently, E. coli was again suspended in 5 ml of cold 1/15 M
potassium phosphate buffer (pH 6.8), subjected to ultrasonic
treatment using a microchip while icing, and centrifuged at
4.degree. C., 15,000.times.g for 20 minutes. The supernatants were
used as crude enzyme solutions.
The crude enzyme solutions were used to determine sensitivity to
carpropamid. The sensitivity to carpropamid was determined in the
same manner as described in Example 1. The results are shown in
FIG. 8. In FIG. 8, the open circles and closed circles represent
the results from determination of sensitivity to carpropamid
measured in Example 1.
Referring to FIG. 8, for both the standard fungus and
carpropamid-resistant fungi, the GST-fused SCDH enzymes expressed
in E. coli exhibited the same drug sensitivity as the SCDH enzyme
contained in the crude enzyme solutions extracted from rice blast
fungi.
Similarly, sensitivity to SCDH inhibitors, fenoxanil and
diclocymet, were also studied. The results are shown in FIG. 9.
Referring to FIG. 9, the GST-fused SCDH enzyme was also found to
show resistance to fenoxanil and diclocymet. In other words, the
results shown in FIGS. 8 and 9 revealed that the GST-fused SCDH
enzyme showed high enzyme activity in the presence of various SCDH
inhibitors. Accordingly, in order to find and/or develop drugs for
preventing and disinfestating for rice blast fungi that exhibit
high infectivity to rice even in the presence of SCDH inhibitors,
candidate agents may be screened using the GST-fused SCDH enzyme.
Specifically, the enzyme activity of the GST-fused SCDH enzyme is
measured in the presence of candidate agents to select a candidate
agent that significantly decreases the enzyme activity. The
selected candidate agent decreases the enzyme activity of the
mutant SCDH enzyme and thus decreases the infectivity of the
resistant rice blast fungus. Accordingly, development of rice blast
caused by resistant rice blast fungi can be prevented.
Example 6
A simple assay for Val75Met mutation in an SCDH enzyme from a rice
blast fungus was considered by applying the PCR-RFLP method.
Similar to Example 3, a rice blast fungus filamentous mycelium was
irradiated with microwaves to simply prepare a genome DNA solution.
Five .mu.l of this genome DNA solution was mixed with 1 .mu.l each
of Primer 8 (SEQ ID NO: 12, 5'-TTCGTCGGCATGGTCTCGAGCATCTAG-3', 25
pmol/.mu.l), Primer 5 (5'-GTGGCCCTTCATGGTGACCTCCT-3', 25
pmol/.mu.l) (SEQ ID NO: 9) and PCR bead (Amersham Bioscience) for a
final volume of 25 .mu.l to prepare a reaction solution.
The underlined bases "TCT" in Primer 8 mismatch the nucleotide
sequence of the genome DNA as a template and are designed to form a
cleavage recognized site ("TCTAGA") for restriction enzyme XbaI
together with the bases "AG" at the 3'-end and the first base that
is amplified by the later-described PCR. When the first base
amplified by the PCR is "A," the fragment amplified by Primer 8
will include the cleavage recognized site for restriction enzyme
XbaI. On the other hand, when the first base amplified by the PCR
is a base other than "A," the cleavage recognized site for
restriction enzyme XbaI is absent in the amplified fragment.
For PCR reaction, 40 cycles of 95.degree. C. for 30 seconds,
55.degree. C. for 1 minute and 72.degree. C. for 2 minute were
repeated. After the final cycle at 72.degree. C. for 7 minutes, the
reaction was carried out and terminated. As a result of this
reaction, a PCR product of 183 bp was obtained. The PCR product was
purified using GFX PCR DNA and the Gel Band Purification Kit
(Amersham Biosciences) and then eluted with a final volume of 20
.mu.l of sterilized water. Of the resultant, 7.5 .mu.l was mixed
with 1 .mu.l.times.M buffer (Takara), 1 .mu.l 10.1% BSA solution
and 0.5 .mu.l XbaI (12 u/.mu.l, Takara) for a final volume of 10
.mu.l and subjected to restriction enzyme reaction at 37.degree. C.
for 1 hour. Results from electrophoresis of the total volume of the
reaction solution in 3% agarose are shown in FIG. 10. In FIG. 10,
Lane 2 represents the reaction solution using the genome DNA
extracted from the standard strain. Lane 3 represents the reaction
solution using the genome DNA extracted from the resistant strain.
Lane 4 represents the reaction solution using the genome DNA
extracted from the standard strain, which had not been subjected to
restriction enzyme reaction. Lane 5 represents the reaction
solution using the genome DNA extracted from the resistant strain,
which had not been subjected to restriction enzyme reaction.
As can be appreciated from FIG. 10, the XbaI-treated sample of the
PCR product from the resistant strain was shorter by about 25
bases. From this result, it became clear that the standard strain
(wild-type strain) and the resistant strain can be distinguished by
applying the PCR-RFLP technique.
INDUSTRIAL APPLICABILITY
As described above, the present invention provides a gene that can
be used extensively, for example, in studies relating to rice blast
fungi resistant to SCDH inhibitors. This gene may be used, for
example, in screening a novel SCDH inhibitor and assessing
sensitivity of a subject rice blast fungus to an SCDH
inhibitor.
SEQUENCE LISTINGS
1
191516DNAPyricularia oryzaeCDS(1)..(516) 1atg ggt tcg caa gtt caa
aag agc gat gag ata acc ttc tca gac tac 48Met Gly Ser Gln Val Gln
Lys Ser Asp Glu Ile Thr Phe Ser Asp Tyr 1 5 10 15ctg ggc ctc atg
act tgc gtc tat gag tgg gca gac agc tac gac tcc 96Leu Gly Leu Met
Thr Cys Val Tyr Glu Trp Ala Asp Ser Tyr Asp Ser 20 25 30aag gac tgg
gat agg ctg cga aag gtc att gcg cct act ctg cgc att 144Lys Asp Trp
Asp Arg Leu Arg Lys Val Ile Ala Pro Thr Leu Arg Ile 35 40 45gac tac
cgc tcc ttc ctc gac aag ctc tgg gag gca atg ccg gcc gag 192Asp Tyr
Arg Ser Phe Leu Asp Lys Leu Trp Glu Ala Met Pro Ala Glu 50 55 60gag
ttc gtc ggc atg gtc tcg agc aag cag atg ctg ggc gac ccc acc 240Glu
Phe Val Gly Met Val Ser Ser Lys Gln Met Leu Gly Asp Pro Thr 65 70
75 80ctc cgc acg cag cac ttc atc ggc ggc acg cgc tgg gag aag gtg
tcc 288Leu Arg Thr Gln His Phe Ile Gly Gly Thr Arg Trp Glu Lys Val
Ser 85 90 95gag gac gag gtc atc ggc tac cac cag ctg cgc gtc ccg cac
cag agg 336Glu Asp Glu Val Ile Gly Tyr His Gln Leu Arg Val Pro His
Gln Arg 100 105 110tac aag gac acc acc atg aag gag gtc acc atg aag
ggc cac gcc cac 384Tyr Lys Asp Thr Thr Met Lys Glu Val Thr Met Lys
Gly His Ala His 115 120 125tcg gca aac ctt cac tgg tac aag aag atc
gac ggc gtc tgg aag ttc 432Ser Ala Asn Leu His Trp Tyr Lys Lys Ile
Asp Gly Val Trp Lys Phe 130 135 140gcc ggc ctc aag ccc gat atc cgc
tgg ggc gag ttc gac ttt gac agg 480Ala Gly Leu Lys Pro Asp Ile Arg
Trp Gly Glu Phe Asp Phe Asp Arg145 150 155 160atc ttt gag gac gga
cgg gag acc ttt ggc gac aaa 516Ile Phe Glu Asp Gly Arg Glu Thr Phe
Gly Asp Lys 165 1702172PRTPyricularia oryzae 2Met Gly Ser Gln Val
Gln Lys Ser Asp Glu Ile Thr Phe Ser Asp Tyr 1 5 10 15Leu Gly Leu
Met Thr Cys Val Tyr Glu Trp Ala Asp Ser Tyr Asp Ser 20 25 30Lys Asp
Trp Asp Arg Leu Arg Lys Val Ile Ala Pro Thr Leu Arg Ile 35 40 45Asp
Tyr Arg Ser Phe Leu Asp Lys Leu Trp Glu Ala Met Pro Ala Glu 50 55
60Glu Phe Val Gly Met Val Ser Ser Lys Gln Met Leu Gly Asp Pro Thr
65 70 75 80Leu Arg Thr Gln His Phe Ile Gly Gly Thr Arg Trp Glu Lys
Val Ser 85 90 95Glu Asp Glu Val Ile Gly Tyr His Gln Leu Arg Val Pro
His Gln Arg 100 105 110Tyr Lys Asp Thr Thr Met Lys Glu Val Thr Met
Lys Gly His Ala His 115 120 125Ser Ala Asn Leu His Trp Tyr Lys Lys
Ile Asp Gly Val Trp Lys Phe 130 135 140Ala Gly Leu Lys Pro Asp Ile
Arg Trp Gly Glu Phe Asp Phe Asp Arg145 150 155 160Ile Phe Glu Asp
Gly Arg Glu Thr Phe Gly Asp Lys 165 1703516DNAPyricularia
oryzaeCDS(1)..(516) 3atg ggt tcg caa gtt caa aag agc gat gag ata
acc ttc tca gac tac 48Met Gly Ser Gln Val Gln Lys Ser Asp Glu Ile
Thr Phe Ser Asp Tyr 1 5 10 15ctg ggc ctc atg act tgc gtc tat gag
tgg gca gac agc tac gac tcc 96Leu Gly Leu Met Thr Cys Val Tyr Glu
Trp Ala Asp Ser Tyr Asp Ser 20 25 30aag gac tgg gat agg ctg cga aag
gtc att gcg cct act ctg cgc att 144Lys Asp Trp Asp Arg Leu Arg Lys
Val Ile Ala Pro Thr Leu Arg Ile 35 40 45gac tac cgc tcc ttc ctc gac
aag ctc tgg gag gca atg ccg gcc gag 192Asp Tyr Arg Ser Phe Leu Asp
Lys Leu Trp Glu Ala Met Pro Ala Glu 50 55 60gag ttc gtc ggc atg gtc
tcg agc aag cag gtg ctg ggc gac ccc acc 240Glu Phe Val Gly Met Val
Ser Ser Lys Gln Val Leu Gly Asp Pro Thr 65 70 75 80ctc cgc acg cag
cac ttc atc ggc ggc acg cgc tgg gag aag gtg tcc 288Leu Arg Thr Gln
His Phe Ile Gly Gly Thr Arg Trp Glu Lys Val Ser 85 90 95gag gac gag
gtc atc ggc tac cac cag ctg cgc gtc ccg cac cag agg 336Glu Asp Glu
Val Ile Gly Tyr His Gln Leu Arg Val Pro His Gln Arg 100 105 110tac
aag gac acc acc atg aag gag gtc acc atg aag ggc cac gcc cac 384Tyr
Lys Asp Thr Thr Met Lys Glu Val Thr Met Lys Gly His Ala His 115 120
125tcg gca aac ctt cac tgg tac aag aag atc gac ggc gtc tgg aag ttc
432Ser Ala Asn Leu His Trp Tyr Lys Lys Ile Asp Gly Val Trp Lys Phe
130 135 140gcc ggc ctc aag ccc gat atc cgc tgg ggc gag ttc gac ttt
gac agg 480Ala Gly Leu Lys Pro Asp Ile Arg Trp Gly Glu Phe Asp Phe
Asp Arg145 150 155 160atc ttt gag gac gga cgg gag acc ttt ggc gac
aaa 516Ile Phe Glu Asp Gly Arg Glu Thr Phe Gly Asp Lys 165
1704172PRTPyricularia oryzae 4Met Gly Ser Gln Val Gln Lys Ser Asp
Glu Ile Thr Phe Ser Asp Tyr 1 5 10 15Leu Gly Leu Met Thr Cys Val
Tyr Glu Trp Ala Asp Ser Tyr Asp Ser 20 25 30Lys Asp Trp Asp Arg Leu
Arg Lys Val Ile Ala Pro Thr Leu Arg Ile 35 40 45Asp Tyr Arg Ser Phe
Leu Asp Lys Leu Trp Glu Ala Met Pro Ala Glu 50 55 60Glu Phe Val Gly
Met Val Ser Ser Lys Gln Val Leu Gly Asp Pro Thr 65 70 75 80Leu Arg
Thr Gln His Phe Ile Gly Gly Thr Arg Trp Glu Lys Val Ser 85 90 95Glu
Asp Glu Val Ile Gly Tyr His Gln Leu Arg Val Pro His Gln Arg 100 105
110Tyr Lys Asp Thr Thr Met Lys Glu Val Thr Met Lys Gly His Ala His
115 120 125Ser Ala Asn Leu His Trp Tyr Lys Lys Ile Asp Gly Val Trp
Lys Phe 130 135 140Ala Gly Leu Lys Pro Asp Ile Arg Trp Gly Glu Phe
Asp Phe Asp Arg145 150 155 160Ile Phe Glu Asp Gly Arg Glu Thr Phe
Gly Asp Lys 165 170521DNAArtificial SequenceDescription of
Artificial Sequence chemically synthesized primer 5gcagtgatac
ccacaccaaa g 21622DNAArtificial SequenceDescription of Artificial
Sequence chemically synthesized primer 6ttatttgtcg gcaaaggtct cc
22744DNAArtificial SequenceDescription of Artificial Sequence
chemically synthesized primer 7agttcgaact ggaattcaac cggcacgcat
gatgcatgca ttta 44821DNAArtificial SequenceDescription of
Artificial Sequence chemically synthesized primer 8atgggttcgc
aagttcaaaa g 21923DNAArtificial SequenceDescription of Artificial
Sequence chemically synthesized primer 9gtggcccttc atggtgacct cct
231020DNAArtificial SequenceDescription of Artificial Sequence
chemically synthesized primer 10acaagctctg ggaggcaatg
201137DNAArtificial SequenceDescription of Artificial Sequence
chemically synthesized primer 11atcgtcgacg tgaattcgtc ttgtaaaagc
cgccaac 371227DNAArtificial SequenceDescription of Artificial
Sequence chemically synthesized primer 12ttcgtcggca tggtctcgag
catctag 2713600DNAPyricularia oryzae 13ctagcaaccg cagtgatacc
cacaccaaag agcttccttc agtctagtat agttcacttc 60aacttgtaaa agccgccaac
atgggttcgc aagttcaaaa gagcgatgag ataaccttct 120cagactacct
gggcctcatg acttgcgtct atgagtgggc agacagctac gactccaagg
180actgggatag gctgcgaaag gtcattgcgc ctactctgcg cattgactac
cgctccttcc 240tcgacaagct ctgggaggca atgccggccg aggagttcgt
cggcatggtc tcgagcaagc 300aggtgctggg cgaccccacc ctccgcacgc
agcacttcat cggcggcacg cgctgggaga 360aggtgtccga ggacgaggtc
atcggctacc accagctgcg cgtcccgcac cagaggtaca 420aggacaccac
catgaaggag gtcaccatga agggccacgc ccactcggca aaccttcact
480ggtacaagaa gatcgacggc gtctggaagt tcgccggcct caagcccgat
atccgctggg 540gcgagttcga ctttgacagg atctttgagg acggacggga
gacctttggc gacaaataaa 60014545DNAPyricularia oryzae 14ctagtatagt
tcacttcaac ttgtaaaagc cgccaacatg ggttcgcaag ttcaaaagag 60cgatgagata
accttctcag actacctggg cctcatgact tgcgtctatg agtgggcaga
120cagctacgac tccaaggact gggataggct gcgaaaggtc attgcgccta
ctctgcgcat 180tgactaccgc tccttcctcg acaagctctg ggaggcaatg
ccggccgagg agttcgtcgg 240catggtctcg agcaagcagg tgctgggcga
ccccaccctc cgcacgcagc acttcatcgg 300cggcacgcgc tgggagaagg
tgtccgagga cgaggtcatc ggctaccacc agctgcgcgt 360cccgcaccag
aggtacaagg acaccaccat gaaggaggtc accatgaagg gccacgccca
420ctcggcaaac cttcactggt acaagaagat cgacggcgtc tggaagttcg
ccggcctcaa 480gcccgacatc cgctggggcg agttcgactt tgacaggatc
tttgaggacg gacgggagac 540ctttg 54515538DNAPyricularia oryzae
15agttcacttc aacttgtaaa agccgccaac atgggttcgc aagttcaaaa gagcgatgag
60ataaccttct cagactacct gggcctcatg acttgcgtct atgagtgggc agacagctac
120gactccaagg actgggatag gctgcgaaag gtcattgcgc ctactctgcg
cattgactac 180cgctccttcc tcgacaagct ctgggaggca atgccggccg
aggagttcgt cggcatggtc 240tcgagcaagc aggtgctggg cgaccccacc
ctccgcacgc agcacttcat cggcggcacg 300cgctgggaga aggtgtccga
ggacgaggtc atcggctacc accagctgcg cgtcccgcac 360cagaggtaca
aggacaccac catgaaggag gtcaccatga agggccacgc ccactcggca
420aaccttcact ggtacaagaa gatcgacggc gtctggaagt tcgccggcct
caagcccgac 480atccgctggg gcgagttcga ctttgacagg atctttgagg
acggacggga gacctttg 53816610DNAPyricularia oryzae 16ctagcaaccg
cagtgatacc cacaccaaag agcttccttc agtctagtat agttcacttc 60aacttgtaaa
agccgccaac atgggttcgc aagttcaaaa gagcgatgag ataaccttct
120cagactacct gggcctcatg acttgcgtct atgagtgggc agacagctac
gactccaagg 180actgggatag gctgcgaaag gtcattgcgc ctactctgcg
cattgactac cgctccttcc 240tcgacaagct ctgggaggca atgccggccg
aggagttcgt cggcatggtc tcgagcaagc 300aggtgctggg cgaccccacc
ctccgcacgc agcacttcat cggcggcacg cgctgggaga 360aggtgtccga
ggacgaggtc atcggctacc accagctgcg cgtcccgcac cagaggtaca
420aggacaccac catgaaggag gtcaccatga agggccacgc ccactcggca
aaccttcact 480ggtacaagaa gatcgacggc gtctggaagt tcgccggcct
caagcccgat atccgctggg 540gcgagttcga ctttgacagg atctttgagg
acggacggga gacctttggc gacaaataaa 600tgcatgcatc
61017732DNAPyricularia oryzae 17tccttcagtc tagtatagtt cacttcaact
tgtaaaagcc gccaacatgg gttcgcaagt 60tcaaaagagc gatgagataa ccttctcagg
tgagcataat atccccctcc aaaaagaaaa 120tagcggtgaa gccaccaacg
acagtaccgc tgaccctaat tcccctccag actacctggg 180cctcatgact
tgcgtctatg agtgggcaga cagctacgac tccaaggact gggataggct
240gcgaaaggtc attgcgccta ctctgcgcgt atgttccgcc ctgccatgtt
tatttttact 300ttcccacacc aaatccagac tttaacagcg acgaccaaaa
aaaaaaaaaa aaaacagatt 360gactaccgct ccttcctcga caagctctgg
gaggcaatgc cggccgagga gttcgtcggc 420atggtctcga gcaagcaggt
gctgggcgac cccaccctcc gcacgcagca cttcatcggc 480ggcacgcgct
gggagaaggt gtccgaggac gaggtcatcg gctaccacca gctgcgcgtc
540ccgcaccaga ggtacaagga caccaccatg aaggaggtca ccatgaaggg
ccacgcccac 600tcggcaaacc ttcactggta caagaagatc gacggcgtct
ggaagttcgc cggcctcaag 660cccgacatcc gctggggcga gttcgacttt
gacaggatct ttgaggacgg acgggagacc 720tttggcgaca aa
73218729DNAPyricularia oryzae 18ttccttcagt ctagtatagt tcacttcaac
ttgtaaaagc cgccaacatg ggttcgcaag 60ttcaaaagag cgatgagata accttctcag
gtgagcataa tatccccctc caaaaagaaa 120atagcggtga agccaccaac
gacagtaccg ctgaccctaa ttcccctcca gactacctgg 180gcctcatgac
ttgcgtctat gagtgggcag acagctacga ctccaaggac tgggataggc
240tgcgaaaggt cattgcgcct actctgcgcg tatgttccgc cctgccatgt
ttatttttac 300tttcccacac caaatccaga ctttaacagc gacgaccaaa
aaaaaaaaaa acagattgac 360taccgctcct tcctcgacaa gctctgggag
gcaatgccgg ccgaggagtt cgtcggcatg 420gtctcgagca agcaggtgct
gggcgacccc accctccgca cgcagcactt catcggcggc 480acgcgctggg
agaaggtgtc cgaggacgag gtcatcggct accaccagct gcgcgtcccg
540caccagaggt acaaggacac caccatgaag gaggtcacca tgaagggcca
cgcccactcg 600gcaaaccttc actggtacaa gaagatcgac ggcgtctgga
agttcgccgg cctcaagccc 660gacatccgct ggggcgagtt cgactttgac
aggatctttg aggacggacg ggagaccttt 720ggcgacaaa 7291933PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide
derived from Pyricularia oryzae 19Gly Asp His Pro Pro Lys Ser Asp
Leu Val Pro Arg Gly Ser Pro Gly1 5 10 15Ile Arg Leu Val Lys Ala Ala
Asn Met Gly Ser Gln Val Gln Lys Ser 20 25 30Asp
* * * * *